Bifunctional compounds which link cytotoxic reagents to antibodies (i.e., xe2x80x9clinkersxe2x80x9d) are known in the art. These compounds have been particularly useful in the formation of immunoconjugates directed against tumor associated antigens. Such immunoconjugates allow the selective delivery of toxic drugs to tumor cells. (See e.g., Hermentin and Seiler, xe2x80x9cInvestigations With Monoclonal Antibody Drug Conjugates,xe2x80x9d Behring Insti. Mitl. 82:197-215 (1988); Gallego et al., xe2x80x9cPreparation of Four Daunomycin-Monoclonal Antibody 791T/36 Conjugates With Anti-Tumor Activityxe2x80x9d. Int. J. Cancer 33:737-44 (1984); Arnon et al., xe2x80x9cIn Vitro and In Vivo Efficacy of Conjugates of Daunomycin With Anti-Tumor Antibodies,xe2x80x9d Immunological Rev. 62:5-27 (1982).
Greenfield et al. have described the formation of acid-sensitive immunoconjugates containing the acylhydrazide conjugated via an acylhydrazone bond to the 13-keto position of an anthracycline molecule, and conjugation of this anthracycline derivative to an antibody molecule (Greenfield et al., European Patent Publication EP 0 328 147, published Aug. 16, 1989, which corresponds to pending U.S. Ser. No. 07/270,509, filed Nov. 16, 1988 and U.S. Ser. No. 07/155,181, filed Feb. 11, 1988, now abandoned). This latter reference also discloses specific thioether-containing linkers and conjugates, including hydrazone thioether containing immunoconjugates.
Kaneko et al. (U.S. Pat. No. 5,137,877 which is equivalent to European Patent Publication, EP A 0 457 250, published Nov. 21, 1991) have also described the formation of conjugates containing anthracycline antibiotics attached to a bifunctional linker by an acylhydrazone bond at the C-13 position of an anthracycline molecule. In their invention the linkers contain a reactive pyridinyidithio- or an ortho-nitrophenyldithio- group, by which the linker reacts with a suitable group attached to a cell reactive ligand, to form the completed conjugate.
Conjugates which rely on simple acid hydrolysis may release the drug prematurely. Accordingly, it would be desirable to have conjugates that release active drug in a more site-specific fashion. European Patent Publication 94107501.2 discloses lysosomal enzymes-cleavable antitumor drug conjugates which are selectively activatible at the site of the tumor. However, one of the problems with prior art immunoconjugates is the relatively low ratio of drug to targeting ligand (e.g., immunoglobulin) achievable. It would be highly desirable to have immunoconjugates, activatible at the tumor site, which provide a higher ratio of drug to targeting ligand.
The present invention provides novel branched peptide linkers. The novel linkers are used to prepare novel drug/linker molecules and biologically active conjugates composed of a targeting ligand, a therapeutically active drug, and a branched peptide linker. The novel conjugates are selectively activatible at the site of of a selected target cell population recognized by the targeting ligand.
As used herein the term xe2x80x9cdrug/linkerxe2x80x9d or xe2x80x9clinker/drugxe2x80x9d refers to the branched peptide linker molecule coupled to two or more therapeutically active drug molecules, and the term xe2x80x9cconjugatexe2x80x9d refers to the drug/linker molecule coupled to the targeting ligand.
The branched peptide linker contains a protein peptide spacer and may also contain a self-immolating spacer which spaces the protein peptide sequence and the drug. The linkers of the invention are branched so that more than one drug molecule per linker are coupled to the targeting ligand. The number of drugs attached to each linker varies by a factor of 2 for each generation of branching. Thus, the number of drug molecules per molecule of linker can be 2, 4, 8, 16, 32, 64, etc. The factor of branching can be expressed mathematically as 2n, wherein n is a positive integer. Thus, a singly branched linker will have a first generation of branching or 21, i.e., contains a potential of two drug molecules per linker. A doubly branched linker will have a second generation of branching or 22, i.e., contains a potential of four drug molecules per linker.
As n rises from n=1, there is a tendency for the solubility of the immunoconjugate to diminish. Solubility can be enhanced by using more water-soluble peptides, or addition of a water-solubilizing moiety such as polyethylene glycol or charged species, e.g., xcex2-alanine, to the drug in such a way that it is released from the drug by either the low pH or the enzymes of the liposomal milieu.
The present invention is directed to a branched peptide linker for linking a thiol group derived from a targeting ligand to two or more drug moieties which comprises a compound having a terminus containing a thiol acceptor for binding to a thiol group (also called a sulfhydryl group) derived from a targeting ligand, at least one point of branching which is a polyvalent atom, such as a carbon atom or a nitrogen atom, allowing for a level of branching of 2n, wherein n is a positive integer, at least two amino acid moieties per branch providing at least one enzymatic site per branch, and at least two other termini containing groups capable of forming covalent bonds with chemically reactive functional groups derived from a drug moiety. It is preferred that n is 1, 2, 3, or 4; more preferably 1, 2, or 3; and most preferably 1 or 2. It is also preferred that the targeting ligand is an antibody or fragment thereof.
As used in the preceeding paragraph, the phrase xe2x80x9cthiol group derived from the targeting ligandxe2x80x9d means that the thiol group is already present on the targeting ligand or that the targeting ligand is chemically modified to contain a thiol group, which modification optionally includes a thiol spacer group between the targeting ligand and the thiol group. Likewise, the phrase xe2x80x9cchemically reactive functional group derived from a drug moietyxe2x80x9d means that the chemically reactive functional group is already present on the drug or the drug is chemically modified to contain such chemically reactive functional group. Such chemically reactive functional groups are groups that are capable of forming covalent bonds with a linker terminus. Examples of such chemically reactive functional groups include primary or secondary amino, hydroxyl, sulfhydroxyl, carboxyli, aldehyde, ketone, and the like.
Also provided by the invention are intermediates for preparing the linkers, drug/linkers, and/or conjugates; and a method for treating or preventing a selected disease state which comprises administering to a patient a conjugate of the invention.
The selected target cell population recognized by the targeting ligand is preferably a tumor.
An aspect of the invention provides tumor-specific conjugates which are highly selective substrates for drug-activating enzymatic cleavage by one or more tumor-associated enzymes.
A further aspect of the invention provides tumor-specific drug conjugates wherein the activating enzyme is one which is present in the tumor in sufficient amounts to generate cytotoxic levels of free drug in the vicinity of the tumor.
Another aspect of the invention provides tumor-specific drug conjugates which are stable to adventitious proteases in blood.
A still further aspect of the present invention provides a tumor-specific conjugate in accordance with the preceding aspects, which is considerably less toxic than the activated drug.
In another aspect the present invention provides methods for delivering the conjugates to target cells in which a modification in biological process is desired, such as in the treatment of diseases such as cancer.
The present invention also provides a method for delivering to the site of tumor cells in a warm-blooded animal an active antitumor drug by administering to said warm-blooded animal the conjugate according to this invention.
The above and other aspects of the present invention are achieved by derivatizing a drug (e.g., an an antitumor agent) linked to a ligand through a peptide linker, made up of a protein peptide sequence and a self-immolating spacer, at a reactive site appropriate for inhibiting the pharmacological activity of the antitumor agent to thereby convert the antitumor agent into a pharmacologically inactive peptidyl derivative conjugate. The peptide linker has at least two amino acid residue sequences specifically tailored so as to render the peptidyl derivatives selective substrates for drug-activating enzymatic cleavage by one or more lysosomal proteases, such as cathepsin B, C, D, or L. The enzymatic cleavage reaction will remove the peptide linker moiety from the drug conjugate and effect release of the antitumor agents in pharmacologically active form selectively at the tumor site. In comparison with ligand-drug linkers which rely on simple acid hydrolysis for drug release this new method provides significantly less systemic toxicity due to premature linker hydrolysis in the blood, consequently a greater amount of the drug is delivered to the tumor site, and the method results in a longer storage life and simplified handling conditions for the conjugate.
The conjugates of the present invention show significantly less systemic toxicity than biparte conjugates and free drug. The conjugates of the invention retain both specificity and therapeutic drug activity for the treatment of a selected target cell population. They may be used in a pharmaceutical composition, such as one comprising a pharmaceutically effective amount of a compound of Formula (III) below, associated with a pharmaceutically acceptable carrier, diluent or excipient.
The present invention is directed to a linker molecule of the formula 
wherein
A is a thiol acceptor;
W is a bridging moiety;
c is an integer of 0 to 1;
a is an integer of 2 to 12;
Q is O, NH, or N-lower alkyl;
p is an integer of 0 or 1;
d is an integer of 0 or 1;
E is a polyvalent atom;
each b is an integer of 1 to 10;
each X is of the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gn
xe2x80x83wherein
Y is two amino acid residues in the L form;
Z is one or two amino acid residues;
m is an integer of 0 or 1;
G is a self-immolative spacer; and
n is a integer of 0 or 1; provided that when n is 0 then xe2x80x94Yxe2x80x94Zm 
is ala-leu-ala-leu or gly-phe-leu-gly;
or each X is of the formula 
wherein each X1 is of the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gn;
and wherein Y, Z, Q, E, G, m, d, p, a, b and n are as defined above;
or each X1 is of the formula 
wherein each X2 is of the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gn;
and wherein Y, Z, G, Q, E, m, d, p, a, b and n are as defined above;
or each X2 is of the formula 
wherein each X3 is of the formula
xe2x80x83xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gn;
and wherein Y, Z, G. Q, E, m, d, p, a, b and n are as defined above;
or each X3 is of the formula 
wherein each X4 is of the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gn;
and wherein Y, Z; G, Q, E, m, d, p, a, b and n are as defined above.
The present invention is also directed to a drug/linker molecule of the formula 
wherein
A is a thiol acceptor;
W is a bridging moiety;
c is an integer of 0 to 1;
a is an integer of 2 to 12;
Q is O, NH, or N-lower alkyl;
p is an integer of 0 or 1;
d is an integer of 0 or 1;
E is a polyvalent atom;
each b is an integer of 1 to 10;
each X is of the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94GnD
xe2x80x83wherein
Y is two amino acid residues in the L form;
Z is one or two amino acid residues;
m is an integer of 0 or 1;
G is a self-immolative spacer;
n is a integer 0 or 1; provided that when m is 0 then xe2x80x94Yxe2x80x94Zmxe2x80x94
is ala-leu-ala-leu (SEQ ID NO:1) or gly-phe-leu-gly (SEQ ID NO:2); and
D is a Drug moiety having a backbone and at least one
chemically reactive functional group pendant thereto chemically reacted to the self-immolative spacer or terminal amino acid residue to form a covalent bond, said functional group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl, carboxyl, aldehyde or ketone;
or each X is of the formula 
wherein each X1 is of the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gnxe2x80x94D;
wherein Y, Z, G, D, Q, E, m, d, p and n are as defined above;
or each X1 is of the formula 
wherein each X2 is of the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gnxe2x80x94D;
and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above;
or each X2 is of the formula 
wherein each X3 is of the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gnxe2x80x94D;
and wherein Y, Z, GI D, Q, E, m, d, p, a, b and n are as defined above;
or each X3 is of the formula 
and wherein each X4 is the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gnxe2x80x94D;
and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above.
Furthermore, the present invention is directed to conjugate of the formula 
wherein
L is a ligand;
q is an integer of 1 to 10;
A is a thiol acceptor;
W is a bridging moiety;
c is an integer from 0 to 1;
a is an integer of 2 to 12;
Q is 0, NH, or N-lower alkyl;
p is an integer of 0 or 1;
d is an integer of 1 or 2;
E is a polyvalent atom;
each b is an integer of 1 to 10;
each X is of the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gnxe2x80x94D
xe2x80x83wherein
Y is two amino acid residues in the L form;
Z is one or two amino acid residues;
m is an integer of 0 or 1;
G is a self-immolative spacer; and
n is a integer of 0 or 1; provided that when n is 0 then xe2x80x94Yxe2x80x94Zmxe2x80x94 is ala-leu-ala-leu (SEQ ID NO:1) or gly-phe-leu-gly (SEQ ID NO:2);
D is a drug moiety having a backbone and at least one chemically reactive functional group pendant thereto reacted to the self-immolative spacer to form a covalent bond, said funtional group selected from the group consisting of a primary or secondary amine, hydroxyl, carboxyl, sulfhydryl, aldehyde, or ketone;
or X is of the formula 
wherein each X1 is of the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gnxe2x80x94D;
and wherein Y, Z, G, D, 0, E, m, d, p, a, b and n are as defined above;
or each X1 is of the formula 
wherein each X2 is of the formula
xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gnxe2x80x94D;
and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above;
or each X2 is of the formula 
wherein each X3 is of the formula
xe2x80x83xe2x80x94COxe2x80x94Yxe2x80x94Zmxe2x80x94Gnxe2x80x94D;
wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above;
or each X3 is of the formula 
wherein end X4 is of the formula
xe2x80x94COxe2x80x94Ymxe2x80x94Zmxe2x80x94Gnxe2x80x94D;
wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above.
As used herein the term xe2x80x9clower alkylxe2x80x9d is an alkyl group having 1 to 3 carbon atoms. It is preferred that the polyvalent atom is carbon or nitrogen.
In one embodiment the drug moiety is an anthracycline antibiotic and the targeting ligand is an antibody.
In a preferred embodiment the anthracycline is bound to the linker at an amino sugar group of the anthriacycline. It is preferred that the sugar moiety is daunosamine. The antibody then is bound, through the linker, to the anthracycline compound. In an especially preferred embodiment, this linkage occurs through a reduced disulfide group (i.e. a free sulfhydryl group (xe2x80x94SH)) on an antibody).
In a most preferred embodiment the anthracycline drug moiety is doxorubicin; the thiol acceptor is a Michael Addition acceptor (from which a Michael Addition Adduct is derived), more preferably is a maleimido- group; and the antibody moiety is a chimeric or humanized antibody, and the point of attachment of the linker to the drug is at the amino group of the sugar moiety of the drug.
The conjugates of the invention retain both specificity and therapeutic drug activity for the treatment of a selected target cell population. They may be used in a pharmaceutical composition, such as one comprising a pharmaceutically effective amount of a compound of Formula (III) associated with a pharmaceutically acceptable carrier, diluent or excipient.
The present invention provides novel drug-linker-ligand conjugates composed of a ligand capable of targeting a selected cell population, and a drug connected to the ligand by a branched peptide linker. The linker contains a thiol acceptor such as a Michael Addition accceptor, a bridging moiety, a point of branching, and a peptide sequence per each branch containing at least two amino acid moieties which provides an enzymatic cleavage site for an enzyme such as cathepsin B, C, D, or L. The branched peptide linker may also contain a self-immolating spacer, which spaces the drug and the protein peptide sequence. It has been discovered that when the peptide seqeunce (i.e., Yxe2x80x94Zm) is ala-leu-ala-leu- (SEQ ID NO:1) or gly-phe-leu-gly (SEQ ID NO:2), then the presence of the self-immolative spacer is optional. The reason that a self-immolative spacer is not required for these peptide sequences is not entirely understood; however, it may be due to response to different enzymes.
The targeting ligand molecule can be an immunoreactive protein such as an antibody, or fragment thereof, a non-immunoreactive protein, or peptide ligand such as bombesin or, a binding ligand recognizing a cell associated receptor such as a lectin, or any protein or peptide that possesses a reactive sulfhydryl-group (xe2x80x94SH) or can be modified to contain such a sulfhydryl group. The thiol acceptor carboxylic is linked to the ligand via a thioether bond, and the drug is linked to the linker via a functional group selected from primary or secondary amine, hydroxyl, sulfhydryl, carboxyl, aldehyde or ketone.
For a better understanding of the invention, the drugs, ligands, peptides and spacers will be discussed individually. The synthesis of the conjugates then will be explained.
It will be understood that in the following detailed description and appended claims, the abbreviations and nomenclature employed are those which are standard in amino acid and peptide chemistry, and that all the amino acids referred to are in the L-form unless otherwise specified.
Some abbreviations used in the present application, unless otherwise indicated, are as follows: AcOH: acetic acid; Alloc: allyloxy-carbonyl; Boc: t-butyloxycarbonyl; DBU: diazobicycloundecene; DCC: dicyclohexylcarbodiimide; DCI: direct chemical ionization; DCU: dicyclohexylurea; DIEA: diisopropylethylamine; DMAP: 4-dimethylaminopyridine; DME: 1,2-dimethoxyethane; DOX: doxorubicin; DTT: dithiothreitol; EEDQ: N-ethoxycarbonyl-2-ethoxy-1;2-dihydroquinoline; EtOAc: ethyl acetate; FAB: fast atom bombardment; Fmoc: fluorenylmethoxycarbonyl; GABA: xcex3-aminobutyric acid; HOBt: N-hydroxybenzotriazole; HRMS: high resolution mass spectroscopy; LDL: low density lipoprotein; MC: 6-maleimidocaproyl; MP: 3-maleimidopropionyl; MPr-BHP: maleimidopropyl-bis-hydroxypropyl; MPr-Mal: maleimidopropyl-malonyl; MEt-IBHE: maleimidoethyl-imino-bis-hydroxyethyl; MMA: mitomycin A, MMC: mitomycin C; Mtr: 4-methoxytrityl; NHS: N-hydroxysuccinimide; NMP: N-methylpyrrolidinone; PABC: p-aminobenzyl-carbamoyl; PAB-OH: p-aminobenzyl alcohol; PNP: p-nitrophenol; TFA: trifluoroacetic acid; THF: tetrahydrofuran.
The peptide linker of the present invention contains two, three or four amino acid residues per branch, together with the self-immolative spacer if present, that provides one or more enzyme cleavage sites. The amino acid moieties collectively form a peptide sequence.
The amino acid residues making up the peptide residues are each selected, independently, from the group of amino acids, preferably naturally occurring amino acids. The naturally occurring amino acids are alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys) methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Preferred naturally occuring amino acids are Ala, Val. Leu, Lys, Ile, Met, Phe, Trp, and Pro. Certain non-naturally occuring amino acids also can be part of the peptide residue. Such non-naturally occuring amino acids include citrulline (Cit) and protected amino acids such as naturally occuring amino acids protected with groups such as acetyl, formyl, tosyl, nitro and the like. When specific amino acids are indicated herein as part of a peptide sequence, they are in the L form unless specified otherwise. The amino acid residues making up the xe2x80x9cYxe2x80x9d moiety must be in the L form. The amino acid residue(s) making up the xe2x80x9cZxe2x80x9d moiety can be either in the L or D form. More preferred amino acids include Lys, Lys protected with acetyl or formyl, Arg, Arg arginine protected with tosyl or nitro groups, His, Orn, Orn protected with acetyl or formyl, Phe, Val, Ala, and Cit. Most preferred are Lys and Cit.
The amino acid residue sequence is specifically tailored so that it will be selectively enzymatically cleaved from the resulting peptidyl derivative drug-conjugate by one or more of the tumor-associated proteases.
The amino acid residue chain length of each branch of the peptide linker preferably ranges from that of a dipeptide to that of a tetrapeptide.
It is preferred that the first amino acid residue (i.e., the first amino acid making up xe2x80x9cYxe2x80x9d, read left to right) is a basic amino acid (e.g., Lys or Arg) or has strong hydrogen bonding capability (e.g., Cit). It is preferred that the second amino acid residue (i.e., the second amino acid making up xe2x80x9cYxe2x80x9d, read left to right) has a hydropholic side chain (e.g., Phe, Val, Ala, Leu or Ile).
The following group of exemplary peptide linker groups, are named in order to illustrate further the conjugates of the present invention:
Phe-Lys, Val-Lys, Phe-Phe-Lys, Lys-Phe-Lys, Gly-Phe-Lys, Ala-Lys,
Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe-Leu-Gly,
Ala-Leu-Ala-Leu, Phe-Ng-tosyl-Arg, Phe-Ng-Nitro-Arg, Lys-Lys,
Lys-Cit, and Cit-Cit.
Specific examples of the preferred embodiment of peptide sequences include Phe-Lys, Val-Lys, Val-Cit, and D-Phe-Phe-Lys.
Numerous specific peptide linker molecules suitable for use in the present invention can be designed and optimized in their selectivity for enzymatic cleavage by a particular tumor-associated protease. The preferred peptide linkers for use in the present invention are those which are optimized toward the protease, cathepsin B, C, D, and L.
The molecules in accordance with the present invention may employ an intermediate self-immolative spacer moiety which spaces and covalently links together the drug moiety and the protein peptide moiety. A self-immolative spacer may be defined as a bifunctional chemical moiety which is capable of covalently linking together two spaced chemical moieties into a normally stable tripartate molecule, releasing one of said spaced chemical moieties from the tripartate molecule by means of enzymatic cleavage; and following said enzymatic cleavage, spontaneously cleaving from the remainder of the molecule to release the other of said spaced chemical moieties. In accordance with the present invention, the self-immolative spacer is covalently linked at one of its ends to the protein peptide moiety and covalently linked at its other end to the chemical reactive site of the drug moiety whose derivatization inhibits pharmacological activity, so as to space and covalently link together the protein peptide moiety and the drug moiety into a tripartate molecule which is stable and pharmacologically inactive in the absence of the target enzyme, but which is enzymatically cleavable by such target enzyme at the bond covalently linking the spacer moiety and the protein peptide moiety to thereby effect release of the protein peptide moiety from the tripartate molecule. Such enzymatic cleavage, in turn, will activate the self-immolating character of the spacer moiety and initiate spontaneous cleavage of the bond covalently linking the spacer moiety to the drug moiety, to thereby effect release of the drug in pharmacologically active form.
In the molecules of Formulas I, II, and III:
G is a self-immolative spacer moiety which spaces and covalently links together the drug moiety and the amino acid, in which the spacer is linked to the drug moiety via the T moiety (as used in the following formulas xe2x80x9cTxe2x80x9d represents a nucleophilic atom which is already contained in the Drug), and which may be represented by the structures of Formulae, (IV), (V), (VI), (VII),or (VIII): 
in which T is O, N or S,
xe2x80x94HNxe2x80x94R1xe2x80x94COTxe2x80x83xe2x80x83Formula (V)
in which T is O, N or S, and
R1 is C1-C5 alkyl; 
(J. Med. Chem., 27: 1447 (1984)
in which T is O, N or S, and
R2 is H or C1-C5 alkyl; 
in which T is O, N or S, 
in which T is O, S or N.
As used herein xe2x80x9cC1-C5 alkylxe2x80x9d is meant to include a branched or unbranched hydrocarbon chain having, unless otherwise noted, one to five carbon atoms, including but not limited to methyl, ethyl, isopropyl, n-propyl, sec-butyl, isobutyl, n-butyl and the like.
A preferred G self-immolative spacer moiety suitable for use in the present invention is PABC represented by the Formula (IVa): 
Another preferred G self-immolative spacer moiety suitable for use in the present invention is GABA represented by the Formula (Va): 
Yet another preferred G self-immolative spacer moiety suitable for use in the present invention is xcex1,xcex1-dimethyl GABA represented by the Formula (Vb): 
Another preferred G self-immolative spacer moiety suitable for use in the present invention is xcex2,xcex2-dimethyl GABA represented by the Formula (Vc): 
In the molecules of Formulas I, II, and III, the thiol acceptor xe2x80x9cAxe2x80x9d is linked to the ligand via a sulfur atom derived from the ligand. The thiol acceptor becomes a thiol adduct after bonding to the ligand through a thiol group via a thioester bond. The thiol acceptor can be, for example, an alpha-substitited acetyl group. Such a group has the formula 
wherein Y is a leaving group. Examples of leaving groups include Cl, Br, I, mesylate, tosylate, and the like. If the thiol acceptor is an alpha-substituted acetyl group, the thiol adduct after linkage to the ligand forms the bond xe2x80x94Sxe2x80x94CH2xe2x80x94
Preferably, the thiol acceptor is a Michael Addition acceptor. A representative Michael Addition acceptor of this invention has the formula 
After linkage the thiol group of the ligand, the Michael Addition acceptor becomes a Michael Addition adduct, such as of the formula A 
wherein L is ligand.
The bridging group ia a bifunctional chemical moiety which is capable of covalently linking together two spaced chemical moieties into a stable tripartate molecule. Examples of bridging groups are described in S. S. Wong, Chemistry of Protein Conjugation and Crosslinking. CRC Press, Florida, (1991); and G. E. Means and R. E. Feeney, Bioconiugate Chemistry, vol. 1, pp.2-12, (1990), the disclosures of which are incorporated herein by reference. Specifically, the bridging group xe2x80x9cWxe2x80x9d covalently links the thiol acceptor to a keto moiety. An example of a bridging group has the formula
xe2x80x83xe2x80x94(CH2)fxe2x80x94(Z)gxe2x80x94(CH2)hxe2x80x94
wherein
f is an integer of 0 to 10,
h is an integer of 0 to 10,
g is an integer of 0 or 1,
provided that when g is 0, then f+h is 1 to 10,
Z is S, O, NH, SO2, phenyl, naphthyl, a polyethylene glycol, a cycloaliphatic hydrocarbon ring containing 3 to 10 carbon atoms, or a heteroaromatic hydrocarbon ring containing 3 to 6 carbon atoms and 1 or 2 heteroatoms selected from O, N, or S.
Preferred cycloaliphatic moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Preferred heteroaromatic moieties include pyridyl, polyethlene glycol (1-20 repeating units), furanyl, pyranyl, pyrimidinyl, pyrazinyl, pyridazinyl, oxazinyl, pyrrolyl, thiazolyl, morpholinyl, and the like.
In the bridging group it is preferred that when g is 0, f+h is an integer of 2 to 6 preferably 2 to 4 and more preferably 2. When g is 1, it is preferred that f is 0, 1 or 2, and that h is 0, 1 or 2.
Preferred bridging groups coupled to thiol acceptors are shown in the Pierce Catalog, pp. E-12, E-13, E-14, E-15, E-16, and E-17 (1992).
The drug conjugates of the present invention are effective for the usual purposes for which the corresponding drugs are effective, and have superior efficacy because of the ability, inherent in the ligand, to transport the drug to the desired cell where it is of particular benefit. Further, because the conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a protein such as tumor necrosis factor.
The preferred drugs for use in the present invention are cytotoxic drugs, particularly those which are used for cancer therapy. Such drugs include, in general, DNA damaging agents, anti-metabolites, natural products and their analogs. Preferred classes of cytotoxic agents include, for example, the enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, the podophyllotoxins, differentiation inducers, and taxanes. Particularly useful members of those classes include, for example, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, carminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere, retinoic acid, butyric acid, N8-acetyl spermidine, camptothecin, and their analogues.
As noted previously, one skilled in the art may make chemical modifications to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
In the conjugate of Formula I,
D is a drug moiety having pendant to the backbone thereof a chemically reactive functional group, by means of which the drug backbone is bonded to the protein peptide linker, said functional group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl, carboxyl, aldehyde or a ketone. Representative of said amino containing drugs are mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N8-acetyl spermidine, 1-(2-chloroethyl)-1,2-dimethanesulfonyl hydraxide, tallysomycin, cytarabine and derivatives thereof.
Representative of said alcohol group containing drugs are etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4-9-diene-2,6-diyne-13-one, (U.S. Pat. No. 5,198,560), podophyllotoxin, anguidine, vincristine, vinblastine, morpholine-doxorubicin, n-(5,5-diacetoxy-pentyl)doxorubicin, and derivatives thereof.
Representative of said sulfhydryl containing drugs are esperamicin and 6-mercaptopurine, and derivatives thereof. Representative of said carboxyl containing drugs are methotrexate, camptothecin (ring-opened form of the lactone), butyric acid, retinoic acid, and derivatives thereof. Representative of said aldehyde and ketone containing drugs are anguidine and anthracyclines such as doxorubicin, and derivatives thereof.
A highly preferred group of cytotoxic agents for use as drugs in the present invention include drugs of the following formulae:

in which
R1 is hydrogen or methyl;
R2 is xe2x80x94NH2, xe2x80x94OCH3, xe2x80x94O(CH2)2OH, xe2x80x94NH(CH2)2SS(CH2)2NHAc, xe2x80x94NHCHxe2x80x94Cxe2x89xa1CH, xe2x80x94NH(CH2)2SS(C6H4)NO2, xe2x80x94O(CH2)2SS(CH2)2OH, xe2x80x94Nxe2x95x90CHxe2x80x94NHOCH3, xe2x80x94NH(C6H4)OH, xe2x80x94NH(CH2)2SS(CH2)2NHCO(CH2)2CH(NH2)COOH 

in which
R1 is hydroxy, amino, C1-C3 alkylamino.
di(C1-C3 alkyI)armino, C4-C6 potymethylene amino, 

in which
R1 is amino or hydroxy;
R2 is hydrogen or methyl;
R3 is hydrogen, fluoro, chloro, bromo or iodo;
R4 is hydroxy or a moiety which complete a salt of the carboxylic acid.




wherein
R2 is hydrogen
R1 is hydrogen or 
xe2x80x83wherein
R3 is NH2, OH, OCH3, NH(C1-C3 akyl) or
N(C1-C3 alkyl)2 
R4 is OH, or NH2,
R5 is methyl or thienyl,
R6 is hydrogen or methyl, or a phosphate salt thereof.
As used herein xe2x80x9c(C1-C3 akyl)xe2x80x9d means a straight or branched carbon chain having from one to three carbon atoms; examples include methyl, ethyl, n-propyl and isopropyl.

in which
R1 is H, CH3 or CHO;
when R2 and R3 are taken singly, R3 is H, and one of R4 and $2 is ethyl and the other is H or OH:
when R2 and R3 are taken together with the carbons to which they are attached, they form an oxirane ring in which case R4 is ethyl;
R5 is hydrogen, (C1-C3 alkyl)xe2x80x94CO, or chlorosubstituted (C1-C2 alkyl)xe2x80x94CO.
As used herein xe2x80x9cC1-C3 alkylxe2x80x9d means a straight or branched carbon chain having from one to three carbon atoms; examples include methyl, ethyl, n-propyl and isopropyl.

in which R1 is a base of one of the formulae: 
in which
R2 is hydrogen, methyl, bromo, fluoro, chloro, or iodo;
R3 is xe2x80x94OH or xe2x80x94NH2;
R4 is hydrogen, bromo, chloro, or iodo.

wherein
R1 is hydroxy;
R2 is hydrogen or hydroxy;
R2 is hydrogen, hydroxy, or acetoxy;
R7 is hydrogen or hydroxy;
R3 is hydrogen, hydroxy, or acetoxy;
R4 is aryl, substituted aryl, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl or t-butoxy;
R5 is C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, or xe2x80x94Zxe2x80x94R6;
Z is a direct bond, C1-6 alkyl or C2-6 alkenyl;
R6 is aryl, substituted aryl, C3-6 cycloalkyl, thienyl or furyl.
As used herein, xe2x80x9calkylxe2x80x9d means a straight or branched saturated carbon chain having from one to six carbon atoms; examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl, sec-butyl, isopentyl, and n-hexyl. xe2x80x9cAlkenylxe2x80x9d means a straight or branched carbon chain having at least one carbon-carbon double bond, and having from two to six carbon atoms; examples include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, and hexenyl. xe2x80x9cAlkynylxe2x80x9d means a straight or branched carbon chain having at least one carbon-carbon triple bond, and from two to six carbon atoms; examples include ethynyl, propynyl, butynyl, and hexynyl. xe2x80x9cArylxe2x80x9d means aromatic hydrocarbon having from six to ten carbon atoms; examples include phenyl and naphthyl. xe2x80x9cSubstituted arylxe2x80x9d means aryl substituted with at least one group selected from C1-6 alkanoyloxy, hydroxy, halogen, C1-6 alkyl, trifluoromethyl, C1-6 alkoxy, aryl, C2-6 alkenyl, C1-6 alkanoyl, nitro, amino, and amido.

wherein
R1 is OH or O;
R2 is HorO;
Anguidine can be targeted at the C-3, C-4, C-8 or C-15 positions, as an ester or hydrazone
H2Nxe2x80x94C6H4xe2x80x94N(CH2CH2Cl)2

wherein
R1 is xe2x80x94CH3, xe2x80x94CH2OH, xe2x80x94CH2OCO(CH2)3CH3 or xe2x80x94CH2OCOCH(OC2H5)2 
R2 is xe2x80x94OCH3, xe2x80x94OH or xe2x80x94H
R3 is xe2x80x94NH2, xe2x80x94NHCOCF3, 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethylamine, 1-cyano-2-methoxyethyl amine, or NHxe2x80x94(CH2)4-CH(OAc)2;
R4 is xe2x80x94OH, xe2x80x94OTHP, or xe2x80x94H; and
R5 is xe2x80x94OH or xe2x80x94H provided that R5 is not xe2x80x94OH when R4 is xe2x80x94OH or xe2x80x94OTHP.
One skilled in the art understands that structural Formula (13) includes compounds which are drugs, or are derivatives of drugs, which have acquired in the art different generic or trivial names. Table I, which follows, represents a number of anthracycline drugs and their generic or trivial names and which are especially preferred for use in the present invention.
Of the compounds shown in Table I, the most highly preferred drug is Doxorubicin. Doxorubicin (also referred to herein as xe2x80x9cDOXxe2x80x9d) is that anthracyclin shown in the formula of Table I in which R1 is xe2x80x94CH2OH, R2 is xe2x80x94OCH3, R3 is xe2x80x94NH2, R4 is xe2x80x94OH, and R5 is xe2x80x94H.
The most highly preferred drugs are taxol, mitomycin C, and the anthracycline antibiotic agents of Formula (13), described previously.
The xe2x80x9cligandxe2x80x9d includes within its scope any molecule that specifically binds or reactively associates or complexes with a receptor or other receptive moiety associated with a given target cell population. This cell reactive molecule, to which the drug reagent is linked via the linker in the conjugate, can be any molecule that binds to, complexes with or reacts with the cell population sought to be therapeutically or otherwise biologically modified and, which possesses a free reactive sulfhydryl (xe2x80x94SH) group or can be modified to contain such a sulfhydryl group. The cell reactive molecule acts to deliver the therapeutically active drug moiety to the particular target cell population with which the ligand reacts. Such molecules include, but are not limited to, large molecular weight proteins such as, for example, antibodies, smaller molecular weight proteins, polypeptides or peptide ligands, and non-peptidyl ligands.
The non-immunoreactive protein, polypeptide, or peptide ligands which can be used to form the conjugates of this invention may include, but are not limited to, transferrin, epidermal growth factors (xe2x80x9cEGFxe2x80x9d), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, tumor growth factors (xe2x80x9cTGFxe2x80x9d), such as TGF-xcex1 and TGF-xcex2, vaccinia growth factor (xe2x80x9cVGFxe2x80x9d), insulin and insulin-like growth factors I and II. Non-peptidyl ligands may include, for example, carbohydrates, lectins, and apoprotein from low density lipoprotein.
The immunoreactive ligands comprise in antigen-recognizing immunoglobulin (also referred to as xe2x80x9cantibodyxe2x80x9d), or an antigen-recognizing fragment thereof. Particularly preferred immunoglobulins are those immunoglobulins which can recognize a tumor-associated antigen. As used, xe2x80x9cimmunoglobulinxe2x80x9d may refer to any recognized class or subclass of immunoglobulins such as IgG, IgA, IgM, IgD, or IgE. Preferred are those immunoglobulins which fall within the IgG class of immunoglobulins. The immunoglobulin can be derived from any species. Preferably, however, the immunoglobulin is of human, murine, or rabbit origin. Furthermore, the immunoglobulin may be polyclonal or monoclonal, preferably monoclonal.
As noted, one skilled in the art will appreciate that the invention also encompasses the use of antigen recognizing immunoglobulin fragments. Such immunoglobulin fragments may include, for example, the Fabxe2x80x2, F(abxe2x80x2)2, Fv or Fab fragments, or other antigen recognizing immunoglobulin fragments. Such immunoglobulin fragments can be prepared, for example, by proteolytic enzyme digestion, for example, by pepsin or papain digestion, reductive alkylation, or recombinant techniques. The materials and methods for preparing such immunoglobulin fragments are well-known to those skilled in the art. See generally, Parham, J. Immunology, 131, 2895 (1983); Lamoyi et al., J. Immunological Methods, 56, 235 (1983); Parham, id., 53, 133 (1982); and Matthew et al., id., 50, 239 (1982).
The immunoglobulin can be a xe2x80x9cchimeric antibodyxe2x80x9d as that term is recognized in the art. Also the immunoglobulin may be a xe2x80x9cbifunctionalxe2x80x9d or xe2x80x9chybridxe2x80x9d antibody, that is, an antibody which may have one arm having a specificity for one antigenic site. such as a tumor associated antigen while the other arm recognizes a different target, for example, a hapten which is, or to which is bound, an agent lethal to the antigen-bearing tumor cell. Altematively, the bifunctional antibody may be one in which each arm has specificity for a different epitope of a tumor associated antigen of the cell to be therapeutically or biologically modified. In any case, the hybrid antibodies have a dual specificity, preferably with one or more binding sites specific for the hapten of choice or more or more binding sites specific for a target antigen, for example, an antigen associated with a tumor, an infectious organism, or other disease state.
Biological bifunctional antibodies are described, for example, in European Patent Publication, EPA 0 105 360, to which those skilled in the art are referred. Such hybrid or bifunctional antibodies may be derived, as noted, either biologically, by cell fusion techniques, or chemically, especially with cross-linking agents or disulfide bridge-forming reagents, and may be comprised of whole antibodies and/or fragments thereof. Methods for obtaining such hybrid antibodies are disclosed, for example, in PCT Application WO83/03679, published Oct. 27, 1983, and published European Application EPA 0 217 577, published Apr. 8, 1987, both of which are incorporated herein by reference. Particularly preferred bifunctional antibodies are those biologically prepared from a xe2x80x9cpolydomaxe2x80x9d or xe2x80x9cquadromaxe2x80x9d or which are synthetically prepared with cross-linking agents such as bis-(maleimido)-methyl ether (xe2x80x9cBMMExe2x80x9d), or with other cross-linking agents familiar to those skilled in the art.
In addition the immunoglobulin may be a single chain antibody (xe2x80x9cSCAxe2x80x9d). These may consist of single chain Fv fragments (xe2x80x9cscFvxe2x80x9d) in which the variable light (xe2x80x9cVLxe2x80x9d) and variable heavy (xe2x80x9cVHxe2x80x9d) domains are linked by a peptide bridge or by disulfide bonds. Also, the immunoglobulin may consist of single VH domains (dAbs) which possess antigen-binding activity. See, eg., G. Winter and C. Milstein, Nature, 349, 295 (1991); R. Glockshuber et al., Biochemistry 29, 1362 (1990); and E. S. Ward et al., Nature 341, 544 (1989).
Especially preferred for use in the present invention are chimeric monoclonal antibodies, preferably those chimeric antibodies having specificity toward a tumor associated antigen. As used herein, the term xe2x80x9cchimeric antibodyxe2x80x9d refers to a monoclonal antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a difference source of species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are especially preferred in certain applications of the invention, particularly human therapy, because such antibodies are readily prepared and may be less immunogenic than purely murine monoclonal antibodies. Such murine/human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding murine immungobulin constant regions. Other forms of chimeric antibodies encompassed by the invention are those in which the class or subclass has been modified or changed from that of the original antibody. Such xe2x80x9cchimericxe2x80x9d antibodies are also referred to as xe2x80x9cclass-switched antibodiesxe2x80x9d. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See, e.g., Morrison, S. L., et al., Proc. Natl Acad. Sci, 81 6851 (1984).
Encompassed by the term xe2x80x9cchimeric antibodyxe2x80x9d is the concept of xe2x80x9chumanized antibodyxe2x80x9d, that is those antibodies in which the framework or xe2x80x9ccomplementarity determining regions (xe2x80x9cCDRxe2x80x9d) have been modified to comprise the CDR of an immunoglobulin of different specificitry as compared to that of the parent immunoglobulin. In a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the xe2x80x9chumanized antibodyxe2x80x9d. See, eg., L. Riechmann et al., Nature 332, 323 (1988); M. S. Neuberger et al.; Nature 314, 268 (1985). Particularly preferred CDR""s correspond to those representing sequences recognizing the antigens noted above for the chimeric and bifunctional antibodies. The reader is referred to the teaching of EPA 0 239 400 (published Sep. 30, 1987), incorporated herein by reference, for its teaching of CDR modified antibodies.
One skilled in the art will recognize that a bifunctional-chimeric antibody can be prepared which would have the benefits of lower immunogenicity of the chimeric or humanized antibody, as well as the flexibility, especially for therapeutic treatment, of the bifunctional antibodies described above. Such bifunctional-chimeric antibodies can be synthesized, for instance, by chemical synthesis using cross-linking agents and/or recombinant methods of the type described above. In any event, the present invention should not be construed as limited in scope by any particular method of production of an antibody whether bifunctional, chimeric, bifunctional-chimeric, humanized, or an antigen-recognizing fragment or derivative thereof.
In addition, the invention encompasses within its scope immunoglobulins (as defined above) or immunoglobulin fragments to which are fused active proteins, for example, an enzyme of the type disclosed in Neuberger, et al., PCT application, WO86/01533, published Mar. 13, 1986. The disclosure of such products is incorporated herein by reference.
As noted, xe2x80x9cbifunctionalxe2x80x9d, xe2x80x9cfusedxe2x80x9d, xe2x80x9cchimericxe2x80x9d (including humanized), and xe2x80x9cbifunctional-chimericxe2x80x9d (including humanized) antibody constructions also include, within their individual contexts constructions comprising antigen recognizing fragments. As one skilled in the art will recognize, such fragments could be prepared by traditional enzymatic cleavage of intact bifunctional, chimeric, humanized, or chimeric-bifunctional antibodies. If, however, intact antibodies are not susceptible to such cleavage, because of the nature of the construction involved, the noted constructions can be prepared with immunoglobulin fragments used as the starting materials; or, if recombinant techniques are used, the DNA sequences, themselves, can be tailored to encode the desired-xe2x80x9cfragmentxe2x80x9d which, when expressed, can be combined in vivo or in vitro, by chemical or biological means, to prepare the final desired intact immunoglobulin xe2x80x9cfragmentxe2x80x9d. It is in this context, therefore, that the term xe2x80x9cfragmentxe2x80x9d is used.
Furthermore, as noted above, the immunoglobulin (antibody), or fragment thereof, used in the present invention may be polyclonal or monoclonal in nature. Monoclonal antibodies are the preferred immunoglobulins, however. The preparation of such polyclonal or monoclonal antibodies now is well known to those skilled in the art who, of course, are fully capable of producing useful immunoglobulins which can be used in the invention. See, e.g., G. Kohler and C. Milstein, Nature 256, 495 (1975). In addition, hybridomas and/or monoclonal antibodies which are produced by such hybridomas and which are useful in the practice of the present invention are publicly available from sources such as the American Type Culture Collection (xe2x80x9cATCCxe2x80x9d) 12301 Parklawn Drive, Rockville, MD. 20852 or, commercially, for example, from Boehringer-Mannheim Biochemicals, P.O. Box 50816, Indianapolis, Ind. 46250.
Particularly preferred monoclonal antibodies for use in the present invention are those which recognize tumor associated antigens. Such monoclonal antibodies, are not to be so limited, however, and may include, for example, the following:
In the most preferred embodiment, the ligand containing conjugate is derived from chimeric antibody BR96, xe2x80x9cChiBR96xe2x80x9d, disclosed in U.S. Ser. No. 07/544,246, filed Jun. 26, 1990 now abandoned and which is equivalent to PCT Published Application, WO 91/00295, published Jan. 10, 1991. ChiBR96 is an internalizing murine/human chimeric antibody and is reactive, ad noted, with the fucosylated Lewis Y antigen expressed by human carcinoma cells such as those derived from breast, lung, colon, and ovarian carcinomas. The hybridoma expressing chimeric BR96 and identified as ChiBR96 was deposited on May 23, 1990, under the terms of the Budapest Treaty, with the American Type Culture Collection (xe2x80x9cATCCxe2x80x9d), 12301 Parklawn Drive, Rockville, Md. 20852. Samples of this hybridoma are available under the accession number ATCC 10460. ChiBR96 is derived, in part, from its source parent, BR96. The hybridoma expressing BR96 was deposited, on Feb. 21, 1989, at the ATCC, under the terms of the Budapest Treaty and is available under the accession number HB 10036. The desired hybridoma is cultured and the resulting antibodies are isolated from the cell culture supernatant using standard techniques now well known in the art. See, e.g., xe2x80x9cMonoclonal Hybridoma Antibodies: Techniques and Applicationsxe2x80x9d, Hurell (ed.) (CRC Press, 1982).
Thus, as used xe2x80x9cimmunoglobulinxe2x80x9d or xe2x80x9cantibodyxe2x80x9d encompasses within its meaning all of the immunoglobulin/antibody forms or constructions noted above.
The linkers, drug/linkers, and conjugates of the invention can be made techniques taught herein, known in the art, or can be made via routine experimentation using as guidance the techniques taught herein and/or known in the art. The attachment of the drug to the linker is accomplished by reacting a nucleophilic atom of the drug (O, N or S) to an electrophilic atom (C, S, P) on either the self-immolating spacer or the carboxy terminus of the peptide. This is illustrated as follows: 
where:
X=leaving group such as Clxe2x88x92, Brxe2x88x92, tosylate, N-hydroxysuccinimide
y=nucleophilic group such as OH, NH2, SH, NH-lower alkyl
R=rest of linker.
Linkage to an aldehyde or ketone can be effected by having the aldehyde or ketone in the form of an enol or N-loweralkyl enamine. It is expected that on release of the free enamine, it will spontaneously hydrolyze to an aldehyde or ketone.
The following reaction schemes are illustrative preparative techiqes (compound numbers corrspond to Example numbers): 